Use of ubiquinol-cytochrome c reductase core protein 2 lactoylated modification as a therapeutic target in the preparation of drugs for treating sepsis cardiomyopathy

Abstract

This invention relates to the field of biomedical technology, and in particular to the application of ubiquitin-cytochrome c reductase core protein 2 lactation modification as a therapeutic target in the preparation of drugs for treating septic cardiomyopathy. This invention discloses the key role of Uqcrc2 K109 lactation modification in SIC and its application as a therapeutic target. Through molecular biology methods such as proteomics analysis, lactomics analysis, and immunoprecipitation, this invention has discovered that Uqcrc2-K109 is a therapeutic target for SIC, and by regulating the lactation level of Uqcrc2-K109, it targets and restores the function of mitochondrial respiratory chain complex III, providing a new strategy for the treatment of SIC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of panthenol-cytochrome c reductase core protein 2 lactation modification as a therapeutic target in the preparation of drugs for treating septic cardiomyopathy. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, with SIC (septic cardiomyopathy) being a major cause of death. Currently, the pathogenesis of SIC is not fully understood, and specific treatments are lacking. Mitochondrial dysfunction in cardiomyocytes is considered one of the core pathological mechanisms of SIC, but its specific regulatory targets remain unclear.

[0003] Lactic acid, a key metabolite accumulating in sepsis, has recently been found to regulate protein function through lactylation. Lactic acidation is a novel post-translational modification (PTM) involved in protein function regulation and disease progression. However, the mechanism of lactylation in sepsis-associated mitochondrial (SIC) remains unclear, especially the relationship between lactylation of mitochondrial-related proteins and SIC, which requires further investigation.

[0004] Uqcrc2 (panthenol-cytochrome c reductase core protein 2) is a core subunit of mitochondrial respiratory chain complex III, involved in electron transport and ATP production. Although Uqcrc2 plays an important role in various diseases, its lactation and function in SIC have not been reported. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides the application of panthenol-cytochrome c reductase core protein 2 lactation modification as a therapeutic target in the preparation of drugs for treating septic cardiomyopathy. The K109 site lactation modification of Uqcrc2 can serve as a therapeutic target for treating septic cardiomyopathy (hereinafter referred to as SIC).

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of lactation modification of the K109 site of panthenol-cytochrome c reductase core protein 2 as a therapeutic target in the preparation of drugs for treating SIC.

[0008] Preferably, SIC is treated by inhibiting lactation at the K109 site of panthenol-cytochrome c reductase core protein 2.

[0009] This invention also provides the application of the K109R mutation of panthenol-cytochrome c reductase core protein 2 in the preparation of drugs for treating SIC.

[0010] The present invention also provides a mutant plasmid for treating SIC, which constructs K109R mutant cardiomyocytes of ubiquitin-cytochrome c reductase core protein 2 via lentiviral transfection technology.

[0011] Preferably, the mutant plasmid protects cardiomyocytes by inhibiting lactation modification at the K109 site of the Uqcrc2 protein and restoring the activity of mitochondrial respiratory chain complex III.

[0012] The present invention also provides a medicament for treating septic cardiomyopathy, the medicament containing an active ingredient that targets and inhibits the lactation of Uqcrc2.

[0013] Preferably, the active ingredient includes a lactate dehydrogenase A inhibitor.

[0014] Preferably, the lactate dehydrogenase A inhibitor comprises sodium oxalate.

[0015] The beneficial effects of this invention are:

[0016] This invention discloses the key role of Uqcrc2 lactation modification in spontaneous intramitochondrial inflammatory disease (SIC) and its application as a therapeutic target. Through proteomics and lactomics analysis, this invention identifies Uqcrc2-K109 as a therapeutic target for SIC and, by regulating the lactation level of Uqcrc2-K109, targets and restores the function of mitochondrial respiratory chain complex III, providing a new strategy for the treatment of SIC. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 To investigate the association between lactation and SIC; (A) colorimetric detection of serum lactate levels in mice (n=6); (B) echocardiographic results of mice in each group (n=6); (C) immunofluorescence detection of lactation levels in myocardial tissue and localization of lactated proteins in organelles; (D) Western blot detection of total lactation (Pan-Kla) expression levels in myocardial tissue (n=3); # Compared with the Sham group, P<0.05;

[0019] Figure 2 Proteomics and lactation analysis in cardiac tissue of SIC; (A) Flowchart of proteomics and lactation screening; (B) Expression heatmap of differentially expressed proteins and lactation sites; (C) Statistical graph of total differentially expressed proteins, lactated proteins and lactation sites; (D) Subcellular structure annotation and classification of differentially lactated proteins; (E) GO enrichment analysis; (F) KEGG pathway enrichment analysis;

[0020] Figure 3To investigate the role of Uqcrc2 lactation in the pathogenesis of SIC; (A) Mass spectrometry analysis to identify the K109 lactation site of Uqcrc2; (B) Crystal structure of Uqcrc2 protein (PDB number: Q9DB77); (C) Alignment of Uqcrc2 sequences from five species; (D) Western blot analysis of total Uqcrc2 protein expression (n=3); (E) Immunoprecipitation analysis of Uqcrc2 protein lactation levels in mouse heart tissue (n=3); # Compared with the Sham group, P<0.05; ns indicates no statistical difference;

[0021] Figure 4 Lactation of Uqcrc2 leads to loss of activity of mitochondrial respiratory chain complex III; (A) Schematic diagram of molecular docking between mitochondrial respiratory chain complex III subunits Uqcrc1 and Uqcrc2; (B) Detection of mitochondrial respiratory chain complex III activity in heart tissue (n=4); # Compared with the Sham group, P<0.05;

[0022] Figure 5 Uqcrc2 protein K109 lactation leads to mitochondrial damage in cardiomyocytes; (A) Cardiomyocyte viability was detected by CCK-8 assay (n=3); (B) Western blot analysis of total lactation (Pan-Kla) expression level; (C) Immunoprecipitation analysis of Uqcrc2 lactation level in cardiomyocytes; (D) Detection of mitochondrial respiratory chain complex III activity in cardiomyocytes (n=4); (E) TMRE analysis of mitochondrial membrane potential (n=6); # Compared with the control group, P<0.05; ns indicates no statistical difference;

[0023] Figure 6 Inhibition of Uqcrc2-K109 lactation can reverse cardiomyocyte damage; (A) Western blot detection of Flag-tagged antibody (n=3); (B) Immunoprecipitation detection of Uqcrc2 lactation level (n=5); (C) Detection of mitochondrial respiratory chain complex III activity in cardiomyocytes (n=5); (D) Dynamic curve of cellular oxygen consumption rate (OCR) showing mitochondrial function; (E) Quantitative analysis of mitochondrial functional parameters in AC16 cells (n=3); (F) TMRE detection of mitochondrial membrane potential (n=6); (G) CCK-8 assay for cell viability (n=8); #Compared with Uqcrc2-WT group, P<0.05; *Compared with Uqcrc2-WT+Nala group, P<0.05;

[0024] Figure 7To demonstrate the effectiveness of sodium oxalate in inhibiting Uqcrc2 lactation and alleviating sepsis-induced leukemia (SIC); (A) Western blot analysis of total lactated (Pan-Kla) protein in cardiac tissue; (B) Immunoprecipitation analysis of Uqcrc2 protein lactation levels in mouse cardiac tissue (n=3); (C) Transmission electron microscopy observation of mitochondrial morphology in the myocardium of each group of mice; (D) Echocardiographic results of each group of mice (n=5); (E) Sepsis scores of each group of mice; (F) Survival curve analysis. #Compared with the Sham group, P<0.05; *Compared with the CLP group, P<0.05;

[0025] Figure 8 Lentiviral vector map for the purpose of targeting. Detailed Implementation

[0026] This invention provides the application of Uqcrc2 lactation modification as a therapeutic target in the preparation of drugs for treating sepsis-induced septicemia (SIC). In this invention, SIC is treated by inhibiting Uqcrc2 lactation. This invention does not impose any particular limitations on the dosage form or preparation method of the drug; those skilled in the art can follow conventional methods. The level of Uqcrc2-K109 lactation in cardiomyocytes of septic mice is significantly increased and directly correlated with decreased activity of mitochondrial respiratory chain complex III.

[0027] This invention also provides the application of the K109R mutation of Uqcrc2 in the preparation of drugs for treating SIC. This invention does not impose any special limitations on the dosage form or preparation method of the drug; those skilled in the art can follow conventional methods.

[0028] This invention provides a mutant plasmid for treating SIC (Small Inflammatory Cardiomyopathy), and constructs cardiomyocytes stably expressing the Uqcrc2 protein K109R mutation using lentiviral transfection technology. In this invention, the active ingredient preferably protects cardiomyocytes by restoring the activity of mitochondrial respiratory chain complex III. This invention does not have specific limitations on the method for constructing the Uqcrc2 protein K109R mutation using lentiviral transfection technology; conventional methods are acceptable.

[0029] In this invention, the Uqcrc2-WT and Uqcrc2-K109R plasmids were constructed by Guangzhou Hanyi Biotechnology Co., Ltd.; the NCBI accession number of Uqcrc2 is NM_003366.4.

[0030] In this invention, a medicament for treating SIC is also provided, wherein the active ingredient preferably comprises a lactate dehydrogenase A inhibitor. In this invention, the lactate dehydrogenase A inhibitor preferably comprises sodium oxalate.

[0031] Sodium oxalate reduces Uqcrc2 lactation levels, restores cardiac function, and significantly improves the survival rate of sepsis models in animals. This invention does not impose specific limitations on the dosage form or preparation method of the drug; those skilled in the art can follow conventional methods.

[0032] To make the present invention more apparent and understandable, preferred embodiments are described in detail below. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available products, and those skilled in the art can follow their instructions.

[0033] In the following embodiments, some of the experimental materials and methods include:

[0034] 1. Grouping of experimental animals and construction of a mouse model of sepsis.

[0035] C57BL / 6 mice, male, 8-10 weeks old, 22-25g, were randomly assigned to the Sham group, CLP group, and CLP + sodium oxalate treatment group.

[0036] (1) CLP group: A mouse model of sepsis was established by cecal ligation and puncture (CLP). The abdomen of anesthetized mice was shaved, and a small incision of less than 1 cm was made to expose the cecum. The ileocecal valve was ligated with 2-0 suture. The cecum was punctured with an 18g needle, and a small amount of feces was squeezed out. After the operation, the cecum was restored to its original position, the wound was sutured with 4-0 suture, and the mice were placed on an electric heating blanket for resuscitation.

[0037] (2) Sham group: The anesthesia method and the surgical procedure were the same as those of the CLP group. After the cecum was turned out, the cecum was not ligated and punctured. The intestine was directly returned to the abdomen and then the skin and muscle layer were sutured.

[0038] (3) In the CLP+sodium oxalate treatment group, sodium oxalate (300-600 mg / kg) was injected intraperitoneally in advance. Based on the results of immunoprecipitation and references, an appropriate sodium oxalate (400 mg / kg) was selected as the concentration for subsequent experiments. Then, the model was constructed according to the method of the CLP group.

[0039] 2. Cell Culture

[0040] AC16 human ventricular myocyte cell line and 293T human embryonic kidney cells (HEK293T) were purchased from Pronosun Biotechnology Co., Ltd. AC16 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin; HEK293T cells were cultured in DMEM medium. All cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0041] 3. Overexpression plasmid packaging of lentivirus

[0042] (1) Genetic information

[0043] Table 1 Gene Information

[0044]

[0045]

[0046] (2) See the target lentiviral vector map. Figure 8 .

[0047] (3) Packaging Lentiviral

[0048] Day 1: Cell Preparation

[0049] ① HEK-293T cells were seeded in 10cm dishes under standard conditions and cultured in a humidified incubator at 37°C with 5% CO2. When the cells were fully adhered and the cell density reached 70%, lentivirus packaging was performed.

[0050] Day 2: Plasmid transfection

[0051] ②Lipofectamine 3000 dilution: Take 41 μL of Lipofectamine 3000 reagent and add 1.5 mL of Opti-MEM. TM Dilute the culture medium, mix thoroughly by pipetting, and label it System A.

[0052] ③ Lentiviral Packaging Mixture: Take 8.3 μL of the target gene plasmid Uqcrc2-WT (concentration 516 ng / ul) or 7.8 μL of Uqcrc2-K109R (concentration 546 ng / ul), along with 5.4 μL of the packaging plasmid psPAX2 (concentration 587 ng / ul) and 2.2 μL of the envelope plasmid pMD2.G (concentration 501 ng / ul), and add them sequentially to 1.5 mL of Opti-MEM. TM Add 35 μL P3000 reagent to the culture medium, mix well by pipetting, and label this as system B.

[0053] ④ Add system A to system B, mix well, and incubate at room temperature for 15 minutes to obtain DNA-liposome complex.

[0054] ⑤ Remove the HEK-293T cell culture dish, discard 50% of the old culture medium, add the above DNA-liposome complex dropwise, return it to a 5% CO2, 37℃ humidified incubator and continue culturing for 8 hours.

[0055] ⑥ Observe the cell state, replace the culture medium containing the transfection system in ⑤ with the packaged culture medium, and put it back into a humidified incubator with 5% CO2 at 37°C for continued culture.

[0056] Days 3-4: Observe fluorescence and collect virus solution.

[0057] ⑦ The plasmid carries the GFP fluorescent reporter gene. At 24 h after transfection, the fluorescence is observed under a fluorescence microscope, and the virus solution is collected and stored at 4°C. The new packaging medium is then replaced. At 52 h after transfection, the fluorescence is observed and the virus solution is collected. The virus solution is mixed with the virus solution collected at 24 h and then filtered through a 0.45 μm filter to obtain the virus solution.

[0058] ⑧ Concentrate the collected virus solution according to the instructions of the virus concentration kit, and then perform MOI determination. The obtained virus solution can be used to infect target cells, or it can be stored at -80℃ for later use.

[0059] 4. Construction of AC16 cells stably expressing Uqcrc2-WT and Uqcrc2-K109R using lentiviruses. Day 1: Cell preparation.

[0060] (1) AC16 cells were seeded into 24-well plates under standard conditions and cultured in a humidified incubator at 37°C with 5% CO2. When the cells were completely adhered to the plate and the cell density reached 50%–70%, lentivirus infection was performed.

[0061] Day 2: Viral Infection

[0062] (2) Take the lentivirus solution out of the -80℃ freezer in advance and thaw it on ice.

[0063] (3) Discard 1 / 2 volume of the original culture medium, add an appropriate volume of virus solution for infection based on the previously determined MOI value, and replenish to the complete culture volume 4 hours after infection. The entire process is carried out in a biosafety cabinet.

[0064] Day 3: Fluid Change

[0065] (4) Discard the culture medium containing the virus, add fresh complete culture medium, and continue to incubate in a humidified incubator with 5% CO2 and 37°C.

[0066] Days 4-7: Observe fluorescence

[0067] (5) The virus contains a GFP reporter gene. The GFP expression efficiency was observed by fluorescence microscopy at 48h and 72h after infection. The GFP expression rate reached more than 90% after 72h of infection.

[0068] Day 7-21: Puromycin Screening

[0069] (6) The peak of GFP expression in the cells was observed. Based on the lowest concentration of puromycin that was previously explored to maximize its killing effect, the medium was changed and passaged in a culture medium containing puromycin. The cells were continuously screened for 14 days. The cell status and survival rate were observed. The cells that survived were the cells that stably expressed the puromycin resistance gene. Samples were collected and the overexpression of the tag antibody was detected by Western blotting. This was the AC16 stable cell line that stably overexpressed Uqcrc2-WT and Uqcrc2-K109R.

[0070] 5. Immunoprecipitation

[0071] (1) Proteins from mouse heart tissue and AC16 cells in Examples 1 and 2 were collected using IP lysis buffer, and protein concentrations were determined by the BCA method.

[0072] (2) Take 500 μg of protein sample, add 1 μg of IP Uqcrc2 antibody, incubate overnight at 4℃, add 25 μL of Protein A / G magnetic beads, incubate at room temperature for 2 h, use a magnetic rack to adsorb the magnetic beads, remove the lysis buffer, wash the magnetic beads 3 times, finally discard the washing buffer, add 80 μL of 1X loading buffer to the magnetic beads, heat in a metal bath for 10 min, then use a magnetic rack to adsorb the magnetic beads and collect the eluent.

[0073] 6. Western blot for protein immunoblotting

[0074] (1) SDS-PAGE preparation: Based on the molecular weight of the target protein, prepare the stacking gel and separating gel according to the SDS-PAGE gel preparation kit (catalog number: NSF12, purchased from Affinity Life Sciences Co., Ltd.). Use the pre-prepared glass plates and gel preparation rack to prepare the gel solution, pour it into the glass plates according to the instructions, and finally insert the sample comb.

[0075] (2) Sample addition: After the gel solidifies, add electrophoresis solution to the electrophoresis tank, remove the comb, and add the sample into the well;

[0076] (3) Protein electrophoresis: The voltage and time for electrophoresis are set according to the molecular weight of the protein.

[0077] (4) Transfer: Activate the PVDF membrane with methanol solution beforehand. After electrophoresis, open the gel casting plate and remove the gel. Place the PVDF membrane and gel in the transfer clamp in the order of positive and negative electrodes, removing any air bubbles between the gel and membrane. Place the transfer clamp into the transfer tank, add the transfer buffer, and set the transfer current and time according to the protein molecular weight. Cool the membrane in an ice bath throughout the transfer process.

[0078] (5) Sealing: After the transfer is completed, the PVDF membrane is placed in 5% skim milk powder and sealed in a shaker at 25°C for 90 minutes.

[0079] (6) Incubation with primary antibody: After blocking, the PVDF membrane was washed 4 times with TBST, 8 min each time. Prepare the appropriate concentration of primary antibody according to the instructions, and then put the PVDF membrane into the diluted primary antibody and incubate overnight at 4°C.

[0080] (7) Incubation of secondary antibody: The primary antibody was retrieved the next day and stored in a refrigerator at 4°C. The membrane was washed 4 times with TBST for 8 minutes each time. Secondary antibody of the corresponding species was prepared using secondary antibody dilution buffer and incubated on a shaker at 25°C for 90 minutes.

[0081] (8) ECL Exposure: Wash the film three times with TBST, 8 min each time. Take ECL chemiluminescence developer and prepare it according to the ratio of A:B = 1:1. Mix well before use. Place the PVDF film on the gel imaging plate, add developer and expose, save the image and analyze the grayscale value using ImageJ software.

[0082] 7. Detection of mitochondrial respiratory chain complex III activity

[0083] (1) The experiment was conducted according to the instructions of the Mitochondrial Respiratory Chain Complex III Activity Assay Kit (catalog number: AKOP007M) purchased from Beijing Box Biotechnology Co., Ltd.

[0084] (2) Take 100 mg of tissue or collect 5 × 10 6 AC16 cells were homogenized thoroughly using a homogenizer or by repeatedly pipetting the cells. Then, the cells were centrifuged at 600g for 10 min at 4°C. The supernatant was transferred to a new EP tube and centrifuged again at 12000g for 15 min at 4°C. The supernatant was discarded, and 200 μL of extraction buffer was added to the precipitate. The cells were then sonicated on ice at 20% power for 5 s, with a 10 s interval, for 15 cycles to obtain the sample for testing. The sample was used to determine the activity and protein concentration of mitochondrial respiratory chain complex III.

[0085] (3) Prepare the reaction system according to the instructions.

[0086] Table 2. Reaction system for detecting the activity of mitochondrial respiratory chain complex III

[0087]

[0088]

[0089] (4) Mix the above system thoroughly, immediately load the 96-well plate onto the microplate reader, and measure the absorbance at 550 nm at 0 min and 2 min. Record the absorbance values ​​of each group as A0 experiment, A0 control, A2 experiment, and A2 control, respectively. Calculate ΔA experiment = A2 experiment - A0 experiment; ΔA control = A2 control - A0 control; and calculate ΔA = ΔA experiment - ΔA control. Determine the sample protein concentration using the BCA method. Calculate the enzyme activity according to the formula in the instruction manual.

[0090] 8. CCK-8 assay for cell viability

[0091] (1) Cell preparation: AC16 cells were seeded in 96-well plates at a density of 8000 cells / well. The cells were divided into groups according to the Nala concentration: 0 μM, 5 μM, 10 μM, 20 μM, 40 μM and 80 μM groups and a blank culture medium group. The cells were cultured overnight in an incubator. When the cell density reached more than 70%, the medium was changed with Nala-containing medium according to the experimental design, and the cells were cultured for another 24 hours.

[0092] (2) CCK-8 detection solution: Prepare CCK-8 detection solution according to the ratio of CCK-8 stock solution: DMEM / F12 = 1:9.

[0093] (3) Detection: Add 100 μL of CCK-8 detection solution to each well, wrap it with tin foil to protect it from light, and incubate it in an incubator.

[0094] (4) Obtain absorbance: Detect absorbance at a wavelength of 450 nm and count the cell survival rate of each group.

[0095] 9. TMRE detection of mitochondrial membrane potential

[0096] (1) Cell preparation: A control group and an exogenous lactic acid (Nala) stimulation experimental group were set up. AC16 cells were seeded in 96-well plates at a density of 70%. The control group was replaced with serum-free medium, and the experimental group was replaced with medium containing 20mM exogenous lactic acid (Nala) for further culture.

[0097] (2) Preparation of TMRE detection solution: TMRE stock solution: detection buffer solution = 1:1000. Prepare the required amount of detection solution, mix thoroughly and set aside.

[0098] (3) Staining: After the treatment time is over, take out the 96-well plate, wash the cells with PBS, discard the PBS solution, add 100 μL of TMRE detection solution to each well, and incubate in the dark for 30 min.

[0099] (4) Washing: In a dark environment, aspirate the TMRE detection solution, wash with buffer solution, aspirate the washing solution, and repeat twice. Finally, add 100 μL of fresh buffer solution to each well, wrap with aluminum foil to protect from light, and observe under a fluorescence microscope within 30 minutes.

[0100] (5) Taking fluorescence images: Under a fluorescence microscope, the mitochondrial membrane potential of each group of cells was observed using the wavelength recommended in the instruction manual (Ex / Em=549 / 575nm), and images were taken. The results were analyzed using ImageJ.

[0101] 10. Seahorse Mitochondrial Stress Test

[0102] (1) Cell preparation: Based on the previously determined suitable cell density, 100 μL of cell suspension was seeded into Seahorse-specific cell culture plates per well, with 3 replicates per group. The wells at the four corners of the culture plate were filled with cell culture medium only and used as blank calibration wells. The plates were first placed in a clean bench for 1 hour to stabilize the cells, and then placed in a cell culture incubator for overnight culture. When the cell density reached 70%, the medium was changed to serum-free medium or medium containing 20 mM exogenous lactate (Nala) according to the group, and cultured for another 24 hours.

[0103] (2) Preparation of materials and instruments: Turn on the Seahorse Analyzer instrument and preheat and equilibrate at 37°C for 24 hours. Add 200 μL of special calibration solution to each well of the Seahorse XF Calibrant plate, hydrate the probe, and place it in a CO2-free incubator at 37°C for 24 hours.

[0104] (3) On the day of the experiment, prepare fresh Seahorse Assay Medium working solution according to the kit instructions and preheat it in a 37°C water bath.

[0105] (4) After 24 hours of cell drug treatment, remove the cell plate from the cell culture incubator, first aspirate 100 μL of the original culture medium in each well, then add 250 μL of preheated Seahorse Assay Medium to each well for rinsing, and aspirate and cycle 3 times. During the process, avoid the pipette tip from directly hitting the bottom of the well. Aspirate and leave a small amount of liquid at the bottom each time to strictly avoid drying out the cells at the bottom. Finally, add 100 μL of Seahorse Assay Medium to each well.

[0106] Place the cell plate in a CO2-free 37°C incubator and allow it to equilibrate for 45 minutes.

[0107] (5) Prepare the Rotenone & antimycin A, Oligomycin and FCCP detection solutions according to the formula described in the instructions.

[0108] (6) Take the Seahorse XF Calibrant plate out of the CO2-free 37°C incubator, install the dosing plate, and inject the Oligomycin, FCCP and Rotenone & antimycin A detection solutions into wells A, B and C of the dosing plate in sequence. Take care to avoid generating air bubbles during the dosing process.

[0109] (7) Open the Seahorse Analyzer instrument-specific analysis software Wave, select the mitochondrial stress test analysis module, enter the grouping information, and click Run.

[0110] (8) As prompted by the instrument, place the Seahorse XF Calibrant combination plate with the added drug into the instrument, and the pH value and oxygen concentration will be automatically detected and corrected.

[0111] (9) After calibration, remove the Seahorse XF Calibrant combination plate and replace it with the cell plate from step (5). Continue running the instrument program. The program will automatically finish running and export the relevant data for subsequent analysis.

[0112] 11. Cardiac function was assessed in each group of mice using echocardiography.

[0113] (1) Mouse preparation: Anesthetize mice with 2.0% isoflurane, then remove hair from the chest area with depilatory cream, and fix them on the ultrasound examination table with their faces up.

[0114] (2) Ultrasound acquisition: After preparation, use a small animal ultrasound machine with a 250MHz probe to acquire B-mode and M-mode images along the long and short axes next to the sternum of the mouse.

[0115] (3) Image analysis: The obtained images are measured and the left ventricular ejection fraction (LVEF) is calculated.

[0116] 12. Transmission electron microscopy observation of mitochondria in heart tissue

[0117] (1) Sample collection: Fresh heart tissue was obtained from the mice in the above groups.

[0118] (2) Initial fixation: After separating the mouse heart tissue, it was quickly placed in a culture dish containing pre-cooled 2.5% glutaraldehyde. A tissue sample was quickly cut from the target location using a freshly opened scalpel blade, with a volume <1 mm. 3 Immediately immerse in pre-cooled 2.5% glutaraldehyde, fix at 4°C for 2-4 hours, then rinse with PBS for 15 minutes each time for 3 times to thoroughly remove any residual 2.5% glutaraldehyde.

[0119] (3) Post-fixation: Transfer the sample to 1% osmium tetroxide solution and fix it at 4°C in the dark for 1-2 hours, then rinse with PBS for 10 minutes × 3 times.

[0120] (4) Dehydration: Use 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol and 100% ethanol in sequence for dehydration, and dehydrate for 15 min × 1 time for each concentration of ethanol.

[0121] (5) Infiltration and embedding: First, soak in a 1:1 mixture of acetone and resin for 2-4 hours, and then soak in pure resin for 12-24 hours.

[0122] (6) Embedding and polymerization: The sample was placed in the embedding mold, fresh resin was injected, and polymerization was carried out in a 60°C drying oven for 48 hours.

[0123] (7) Ultrathin slices

[0124] Trim the embedding block to expose the target area. Then, use an ultramicrotome to cut 50-70 nm thick sections. Subsequently, use a toothpick to transfer the sections onto a copper grid coated with a carbon film and allow them to dry at room temperature.

[0125] (8) Staining

[0126] Uranium acetate staining: Float a copper mesh on uranium acetate staining solution (2% aqueous solution) and stain in the dark for 15-30 minutes. Rinse three times with ultrapure water and blot dry with filter paper.

[0127] Lead citrate staining: Float a copper mesh on the lead citrate staining solution and stain for 5-10 minutes (place NaOH particles around the staining solution to avoid CO2 interference). Rinse thoroughly with ultrapure water and dry before use.

[0128] (9) Transmission electron microscopy observation

[0129] Mount the copper mesh onto the electron microscope sample holder and insert it into the electron microscope chamber. Adjust the accelerating voltage (80-120kV), focus, and locate the target region. Observe mitochondrial characteristics: double membrane structure, smooth outer membrane, inner membrane folded inward to form cristae; matrix granules, dense granules that may be present in the mitochondrial matrix. Note the distinction from structures such as the endoplasmic reticulum and lysosomes (mitochondrial cristae are a typical feature).

[0130] (10) Take pictures and save the data.

[0131] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0132] Example 1

[0133] Identification of lactation in Uqcrc2-K109

[0134] Sepsis model mice and control mice were obtained, and serum lactate levels were measured by colorimetric method. Results showed that serum lactate levels were significantly increased in the CLP group (P<0.05). Figure 1 (A). Echocardiography results showed that the left ventricular ejection fraction (LVEF) was significantly decreased in the CLP group mice. Figure 1 The presence of B in the CLP group indicated impaired cardiac function in the septic mice. Immunofluorescence assays showed that cardiomyocytes in the CLP group were swollen and hypertrophied, with partial breakage of cardiomyocyte fibers. Furthermore, the expression level of lactated protein was elevated in cardiac tissue samples from the CLP group, and it was expressed in both the nucleus and cytoplasm. Figure 1(C). Immunoblotting analysis also showed that the expression level of pan-lysine lactation (Pan-Kla) in cardiac tissue was significantly increased in the CLP group. Figure 1 (D). Heart tissues from two groups of mice were collected for 4D proteomics and lactomemography analysis. The results showed that, using a fold change (|Fold change|>1.5) between the CLP group and the Sham group as the threshold, proteomics identified 232 differentially expressed proteins, while lactomemography revealed 439 differentially expressed lactate modification sites in 177 proteins. Figure 2 (B) Compared with the Sham group, the CLP group showed increased expression at 431 lactation modification sites on 169 proteins, and decreased expression at 8 lactation modification sites on 8 proteins. Figure 2 (C). Furthermore, we performed subcellular structural localization annotation and classification of differentially expressed modification sites, and the results showed that the vast majority of lactation-modified proteins (47.67%) were located in mitochondria (…). Figure 2 (D). In the CLP group, the Uqcrc2-K109 lactation level was 7.14 times higher than that in the Sham group, and Uqcrc2 is involved in multiple important pathways such as oxidative stress, oxidative phosphorylation, and diabetic cardiomyopathy. Figure 2 Mass spectrometry analysis confirmed the K109 lactation site (A in section 3) of Uqcrc2, and this site is evolutionarily conserved across multiple species. Figure 3 (C). This invention validates the lactation modification of Uqcrc2 using immunoprecipitation and Western blot experiments with the IP antibody Uqcrc2. In vivo experiments revealed no significant difference in the expression level of Uqcrc2 protein in the heart tissues of mice in the Sham and CLP groups. Figure 3 In the CLP group, the Uqcrc2 lactation level was significantly higher than that in the Sham group (P<0.05). Figure 3 (E).

[0135] Example 2

[0136] Effects of Uqcrc2 lactation modification on mitochondrial respiratory chain complex III activity, mitochondrial membrane potential (TMRE), and cardiomyocyte viability in cardiomyocytes

[0137] This invention illustrates the effects of Uqcrc2 lactation on mitochondrial function and cell viability by detecting mitochondrial respiratory chain complex III activity, mitochondrial membrane potential, and cell viability in cardiomyocytes. The results showed that, compared with the Sham group, the CLP group had significantly lower mitochondrial respiratory chain complex III activity in cardiac tissue. Figure 4 (B)

[0138] In in vitro experiments, exogenous lactic acid stimulation was found to significantly decrease cardiomyocyte activity. Figure 5 (A) Exogenous lactate induced a significant increase in the lactation level of Uqcrc2 protein in cardiomyocytes. Figure 5 (C). Simultaneously, the activity of mitochondrial respiratory chain complex III in cardiomyocytes significantly decreased ( Figure 5 (D). Moreover, exogenous lactate stimulation can lead to disruption of mitochondrial membrane potential, and the difference is statistically significant (P<0.05). Figure 5 (E).

[0139] Example 3

[0140] The K109R mutant plasmid of Uqcrc2 (where the 109th amino acid of the protein is mutated from K to R) exerts a cardioprotective effect by inhibiting lactation at the K109 site of the Uqcrc2 protein.

[0141] This invention mutates the Uqcrc2-K109 site from K to R (lysine to arginine) to mimic the delactated state of the protein. AC16 cardiomyocytes were transfected with a lentiviral vector overexpressing the wild-type Uqcrc2-WT plasmid (Uqcrc2-WT group) and the mutant Uqcrc2-K109R plasmid (Uqcrc2-K109R group). Western blot analysis showed no significant difference in Flag expression between the two groups. Figure 6 (A). Simultaneously, immunoprecipitation results showed that, compared to the Uqcrc2-WT group, the Uqcrc2-K109R group had a significantly lower level of Uqcrc2 lactation modification (A). Figure 6 (B) indicates that the Uqcrc2-K109R mutant leads to delactation of Uqcrc2, and the K109 site is a key lactation site of Uqcrc2.

[0142] This invention reveals that the K109R mutant plasmid exerts a crucial influence on the activity of mitochondrial respiratory chain complex III and mitochondrial membrane potential in cardiomyocytes by downregulating Uqcrc2-K109 lactation. Results show that, compared with the Uqcrc2-WT+Nala group, the mitochondrial respiratory chain complex III activity in the Uqcrc2-K109R+Nala group was significantly restored. Figure 6 (C) 。 The SeahorseXF mitochondrial stress test revealed that exogenous lactate stimulation impaired mitochondrial respiratory chain function, while mutation at the Uqcrc2-K109 site significantly restored mitochondrial respiratory chain function impairment caused by exogenous lactate stimulation. Figure 6 (DE). The mitochondrial membrane potential of the Uqcrc2-K109R+Nala group was significantly restored ( Figure 6 (Middle F). Meanwhile, the cell survival rate of the Uqcrc2-K109R+Nala group was significantly increased ( Figure 6 (G).

[0143] Example 4

[0144] Sodium oxalate's therapeutic effect on SCI by regulating Uqcrc2 lactation

[0145] Animal experiments showed that sodium oxalate dose-dependently inhibited the level of Uqcrc2 lactation in cardiac tissue ( Figure 7 In the CLP + sodium oxalate (400 mg / kg) treatment group, the Uqcrc2 lactation level was significantly reduced by 44% compared to the CLP group. Figure 7 In the middle B), the left ventricular ejection fraction (LVEF) was significantly increased. Figure 7 (D). Transmission electron microscopy of mouse heart tissue in each group showed that mitochondrial cristae were broken down and dissolved in the CLP group, while mitochondrial damage was reduced in the CLP + sodium oxalate treatment group. Simultaneously, sodium oxalate treatment reduced the sepsis severity score in mice and demonstrated good survival rates. Figure 7 (E and F in the middle).

[0146] As can be seen from the above embodiments, this invention reveals for the first time the crucial role of Uqcrc2 lactation modification in SIC and provides a therapeutic strategy targeting this target. Experimental data show that inhibiting Uqcrc2 lactation can significantly improve mitochondrial function and cardiac function, laying a scientific foundation for drug development in SIC.

[0147] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a plasmid overexpressing the K109R mutant of panthenol-cytochrome c reductase core protein 2 in the preparation of a drug for treating septic cardiomyopathy, characterized in that, The plasmid overexpressing the K109R mutant of ubiquitin-cytochrome c reductase core protein 2 protects cardiomyocytes by restoring mitochondrial respiratory chain complex III activity by inhibiting lactation modification at the K109 site of ubiquitin-cytochrome c reductase core protein 2; the NCBI accession number for ubiquitin-cytochrome c reductase core protein 2 is NM_003366.

4. The K109R mutant is a mutant in which the 109th amino acid of the core protein 2 of panthenol-cytochrome c reductase is changed from K to R.

2. A plasmid for treating septic cardiomyopathy, characterized in that, The plasmid is a K109R mutant overexpressing ubiquitin-cytochrome c reductase core protein 2; the NCBI accession number for ubiquitin-cytochrome c reductase core protein 2 is NM_003366.

4. The K109R mutant is a mutant in which the 109th amino acid of the core protein 2 of panthenol-cytochrome c reductase is changed from K to R.

3. The plasmid according to claim 2, characterized in that, The plasmid protects cardiomyocytes by restoring the activity of mitochondrial respiratory chain complex III by inhibiting lactation modification at the K109 site of panthenol-cytochrome c reductase core protein 2.

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